Yearly, influenza virus causes approximately 500,000 deaths (Brown et al. 2009, Immunology and Cell Biology 87, 300-308). With the emergence of a novel viral subtype, deaths can rise into the millions. Id. For example, the pandemic of 1918-1919 killed more than 40 million people, and this was when rapid air travel was much less common (Doherty et al. 2008, The Journal of Clinical Investigation 118, 3273-3275).
Given the fact that the hemagglutinin surface proteins (HA) exist in 16 subtypes, the neuraminidase (NA) in nine subtypes, and the potential for recombination exists in the animal kingdom as well as in the human, many potential pandemic influenza A virus candidates exist. Once an influenza virus has become a seasonal virus, usually after a pandemic, it is going to drift or change over time. Several seasonal viruses are presently in co-circulation: An influenza A virus of the subtype H3N2, and another of the subtype H1N1, and two influenza virus type B strains from the Yamagata and the Victoria lineages. After the recent swine flu pandemic, the new variant of the influenza A H1N1 subtype (vH1N1 or H1N1 new) became the new seasonal H1N1 strain.
In any case, approaches for pandemic influenza vaccines, as well as seasonal influenza vaccines, warrant a combination of several influenza viruses. For pre-pandemic vaccines, a combination of several strains into one vaccine candidate is indicated to either prime against several viruses simultaneously in a pre-pandemic setting or to limit a stockpile to a few vaccines (vaccine library), but each vaccine with a multivalent option, i.e. being protective against several strains to increase its potential.
At present, seasonal vaccines should cover at least three strains, two A-strains and one B-strain.
In this way, the concept of multivalency is motivated by different reasons with regard to pandemic versus seasonal vaccines, but practically leading to similar vaccine construct approaches.
For priming in naïve populations, for a pandemic vaccine generally the entire population and for a seasonal vaccine the young children, an induction of immunity as similar as possible to the wild virus infection in regard to internal and external antigens is desired. For boosting vaccination in subjects already primed by either a wild type influenza (sometimes also called “flu” herein) infection or a flu vaccination, pre-existing immunity should not prohibit a sufficient booster response. Priming can consist of one or several doses (a priming schedule) and boosting most often of only one vaccination.
The principal mechanism of action of current subunit or inactivated, detergent-disrupted influenza virus vaccines is to induce neutralizing antibodies (Doherty et al. 2008, The Journal of Clinical Investigation 118, 3273-3275). Commonly used inactivated seasonal influenza vaccines induce protective antibody responses against the immunizing virus strains (Brown et al. 2009, Immunology and Cell Biology 87, 300-308). However, the antibody response may not be effective against novel virus strains. Id. Antigenic drift occurs in both type A and type B influenza and results in neutralization-resistant mutants. Id. Thus, it is necessary to constantly produce new vaccines to combat these new strains.
MVA (modified vaccinia virus Ankara) originates from the dermal vaccinia virus strain chorioallantois vaccinia virus Ankara (CVA) that was maintained in the Vaccination Institute, Ankara, Turkey for many years and used for vaccination of humans. Due to the often severe post-vaccinal complications associated with vaccinia viruses, there were several attempts to generate a more attenuated, safer smallpox vaccine.
During the period of 1959 to 1974, Prof. Anton Mayr succeeded in attenuating CVA by over 570 continuous passages in CEF cells (Mayr A and Munz E 1964, Veränderung von Vaccinevirus durch Dauerpassagen in Hühnerembryofibroblasten-Kulturen, Zentralbl. Bakteriol. 195, 24-35; Mayr A, Hochstein-Mintzel V, Stickl H 1975, Passage History: Abstammung, Eigenschaften and Verwendung des attenuierten Vaccina-Stammes MVA. Infection 3, 6-14). As part of the early development of MVA as a pre-smallpox vaccine, there were clinical trials using MVA-517 (corresponding to the 517th passage) in combination with Lister Elstree (Stickl H A 1974, Smallpox vaccination and its consequences: first experiences with the highly attenuated smallpox vaccine “MVA”. Prev. Med. 3[1], 97-101; Stickl H & Hochstein-Mintzel V 1971, Intracutaneous smallpox vaccination with a weak pathogenic vaccinia virus (“MVA virus”). Munch. Med. Wochenschr. 113, 1149-1153) in subjects at risk for adverse reactions from vaccinia. In 1976, MVA derived from MVA-571 seed stock (corresponding to the 571st passage) was registered in Germany as the primer vaccine in a two-stage parenteral smallpox vaccination program. Subsequently, MVA-572 was used in approximately 120,000 Caucasian individuals, the majority children between 1 and 3 years of age, with no reported severe side effects, even though many of the subjects were among the population with high risk of complications associated with vaccinia (Mayr et al. 1978, Der Pockenimpfstamm MVA: Marker, genetische Struktur, Erfahrungen mit der parenteralen Schutzimpfung and Verhalten im abwehrgeschwächten Organismus. Zbl. Bakt. Hyg., I. Abt. Orig. B 167, 375-390). MVA-572 was deposited at the European Collection of Animal Cell Cultures as ECACC V94012707.
Being that many passages were used to attenuate MVA, there are a number of different strains or isolates, depending on the passage number in CEF cells. All MVA strains originate from Dr. Mayr and most are derived from MVA-572 that was used in Germany during the smallpox eradication program, or MVA-575 that was extensively used as a veterinary vaccine. MVA-575 was deposited on Dec. 7, 2000, at the European Collection of Animal Cell Cultures (ECACC) with the deposition number V00120707.
By serial propagation (more than 570 passages) of the CVA on primary chicken embryo fibroblasts, the attenuated CVA-virus MVA (modified vaccinia virus Ankara) was obtained. MVA was further passaged by Bavarian Nordic and is designated MVA-BN, corresponding to passage 583. MVA as well as MVA-BN, lacks approximately 13% (24.5 kb from six regions) of the genome compared with ancestral CVA virus (Meisinger-Henschel et al., 2007, Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara. J. Gen. Virol. 88, 3249-3259). The deletions affect a number of virulence and host range genes, as well as the gene for type A inclusion bodies. A sample of MVA-BN was deposited on Aug. 30, 2000 at the European Collection of Cell Cultures (ECACC) with the deposition number V00083008.
MVA-BN can attach to and enter human cells where virally-encoded genes are expressed very efficiently. However, assembly and release of progeny virus does not occur. Preparations of MVA-BN and derivatives have been administered to many types of animals, and to more than 2000 human subjects, including immunodeficient individuals. All vaccinations have proven to be generally safe and well tolerated.
The perception from many different publications is that all MVA strains are the same and represent a highly attenuated, safe, live viral vector. However, preclinical tests have revealed that MVA-BN demonstrates superior attenuation and efficacy compared to other MVA strains (Chaplin P J, Howley P, Meisinger C 2002, Modified Vaccinia Ankara Virus Variant. International Patent Application WO 02/42480). MVA-BN has been shown to have the highest attenuation profile compared to other MVA strains and is safe even in severely immunocompromised animals.
Although MVA exhibits strongly attenuated replication in mammalian cells, its genes are efficiently transcribed and translated, with the block in viral replication being at the level of virus assembly and egress. (Sutter and Moss 1992, Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc. Natl. Acad. Sci. U.S.A 89, 10847-10851; Carroll and Moss 1997, Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238, 198-211.) Despite its high attenuation and reduced virulence, in preclinical studies MVA has been shown to elicit both humoral and cellular immune responses to vaccinia virus proteins and the products of genes cloned into the MVA genome (Harrer et al. 2005, Therapeutic Vaccination of HIV-1-infected patients on HAART with recombinant HIV-1 nef-expressing MVA: safety, immunogenicity and influence on viral load during treatment interruption. Antiviral Therapy 10, 285-300; Cosma et al. 2003, Therapeutic vaccination with MVA-HIV-1 nef elicits nef-specific T-helper cell responses in chronically HIV-1 infected individuals. Vaccine 22(1), 21-29; Di Nicola et al. 2003, Clinical protocol. Immunization of patients with malignant melanoma with autologous CD34(+) cell-derived dendritic cells transduced ex vivo with a recombinant replication-deficient vaccinia vector encoding the human tyrosinase gene: a phase I trial. Hum. Gene Ther., 14[14], 1347-1360; Di Nicola et al. 2004, Boosting T cell-mediated immunity to tyrosinase by vaccinia virus-transduced, CD34(+)-derived dendritic cell vaccination: a phase I trial in metastatic melanoma. Clin. Cancer Res. 10[16], 5381-5390.)
MVA-BN and recombinant MVA-BN-based vaccines can be generated, passaged, produced and manufactured in CEF cells cultured in serum-free medium. Many recombinant MVA-BN variants have been characterized for preclinical and clinical development. No differences in terms of the attenuation (lack of replication in human cell lines) or safety (preclinical toxicity or clinical studies) have been observed between MVA-BN, the viral vector backbone, and the various recombinant MVA-based vaccines.
The safety and immunogenicity of MVA-BN and recombinant MVA-BN vaccines have been demonstrated in more than 15 completed or on-going clinical trials in healthy subjects, patients diagnosed with atopic dermatitis, HIV infected patients, and cancer (melanoma) patients.
A recombinant MVA expressing influenza virus hemagglutinin (HA) and nucleoprotein (NP) genes was generated and tested in mice. Antibody and CTL responses were generated and the mice were protected against a lethal challenge of influenza virus (Sutter et al. 1994, Vaccine 194, 1032-1040).
An H5N1 vaccine candidate based on the replication-deficient modified vaccinia virus Ankara (MVA) has been generated and tested in mice (Kreijtz et al. 2007, Journal of Infectious Diseases 195, 1598-1606). The MVA expressed the hemagglutinin (HA) gene from influenza virus A/Hongkong/156/97 (MVAHA-HK/97) or ANietnam/1194/04 (MVA-HA-VN/04). Id. The mice were then challenged with 3 antigenically distinct strains of H5N1 influenza viruses: A/Hongkong/156/97, ANietnam/1194/04, and A/Indonesia/5/05. Id. A 2-dose immunization regimen induced strong antibody responses that partially cross-reacted with heterologous H5N1 strains. Id. The elicited antibody responses correlated with protection against challenge infection with homologous and heterologous influenza virus strains. Id. Similarly, immunization of macaques with MVA-HA-VN/04 induced (cross-reactive) antibodies and prevented virus replication in the upper and lower respiratory tract and the development of severe necrotizing bronchointerstitial pneumonia (Kreijtz et al. 2009, Vaccine 27, 6296-6299).
A vaccinia-based influenza vaccine, which expresses the immune stimulatory cytokine IL-15, the hemagglutinin, neuraminidase, and nucleoprotein derived from the H5N1 influenza virus ANietnam/1203/2004, and the matrix proteins M1 and M2 from the H5N1 A/CK/Indonesia/PA/2003 virus on the backbone of a currently licensed smallpox vaccine, was generated (Poon et al. 2009, Journal of Immunology 182, 3063-3071). The vaccine induced cross-neutralizing antibodies and cellular immune responses in vaccinated mice and conferred sterile cross-Glade protection when challenged with an H5N1 virus of a different Glade. Id.
There are number of problems with the currently marketed seasonal flu vaccines. First, yearly adaptation of strains is required according to forecast by WHO, with a short window to immunize target populations. This is especially true for young children, who require two doses for priming. The standard vaccine, killed trivalent-split/subunit (TIV), is poorly priming in children. That is, it induces no or only weak immunity in naïve individuals, which is suspected to compromise induction of cross-immunity to other strains/subtypes and thus being counterproductive to the immune constitution (Bodewes et al. 2009, Lancet 9, 784-788; Bodewes et al. 2009, PlosOne 9, e5538:1-9)
It is also poorly immunogenic in elderly persons. An adjuvanted TIV induces stronger immunity in children and elderly, but the impact on immune constitution in young children is unknown. Another vaccine, cold-adapted live-attenuated (CAIV) is a good priming vaccine in naïve subjects, but is a poorly boosting vaccine in pre-immune subjects, e.g. adults. Due to safety concerns in young children, asthmatic attacks and increased incidence of hospitalization after vaccination, it is licensed only for healthy children over two years of age and for asthmatic children over five years of age.
Based on the above, there is a need in the art for flu vaccines both for pandemic flu and for seasonal flu, particularly in young children, and providing increased protection for older children and adults. The current invention fulfills this need and provides means and methods for combating flu by modified vaccinia virus Ankara (MVA)-based vectors as vaccines. The MVA-based vectors build a platform that allows fast and efficient production of flu vaccines that are preferably envisaged not to be subject of adaptation on a yearly basis because of the multivalency of the vaccine and its potential cross-protection. This is achieved by the choice of the external and internal influenza virus antigens and/or an antigenic determinant or epitope thereof offered to the immune system by vaccination.